Annual Research & Review in Biology 36(6): 46-54, 2021; Article no.ARRB.69680 ISSN: 2347-565X, NLM ID: 101632869 Occurrence, Distribution and Identification of Viruses Infecting Some Cucurbits Across Major Cucurbit-Growing Areas in Cross River State, Nigeria O. I. Eyong1*, E. E. Ekpiken2 and O. A. Iso2 1Department of Forestry Wildlife Managment, Cross River University of Technology, Calabar, Nigeria. 2Department of Plant and Biotechnology, Cross River University of Technology, Calabar, Nigeria. Authors’ contributions This work was carried out in collaboration among all authors. Author OIE designed the study, wrote the protocol and wrote the first draft of the manuscript. Authors EEE managed the analyses of the study. Author OAI read the first draft of the manuscript. All authors read and approved the final manuscript Article Information DOI: 10.9734/ARRB/2021/v36i630389 Editor(s): (1) Dr. Manikant Tripathi, Dr. Ram Manohar Lohia Avadh University, India. Reviewers: (1) Justus Eronmosele Omijeh, Modibbo Adama University of Technology,Yola, Nigeria. (2) J. O. Cheikyula, Joseph Sarwuan Tarka University, Nigeria. Complete Peer review History: http://www.sdiarticle4.com/review-history/69680 Received 06 April 2021 Original Research Article Accepted 13 June 2021 Published 23 June 2021 ABSTRACT Cucurbits are group of plants which belonged in the Cucurbitaceae family and constitute one of the most important vegetables for human consumption throughout the world. A survey conducted between January to March 2021 across major cucurbit-growing area in Cross River State revealed wide spread virus infection of these crops, surveyed plants showed mosaic, mottle and leaf deformation symptoms. This research was aimed at identifying viruses infecting some cucurbits in the study areas. Twenty four samples showing virus like symptoms were collected and tested against ACP-ELISA and RT-PCR. Result obtained from ACP-ELISA showed that 20 out of 24 samples collected tested positive to universal potyvirus antisera, the gene sequence analysis revealed that samples were predominantly infected with Algerian watermelon mosaic virus representing 40% of total viruses detected followed by Morroccan watermelon mosaic virus representing 15% of total viruses detected followed by Papaya ringspot virus, Zucchini tigre mosaic virus, Watermelon mosaic virus and Soybean mosaic virus representing 10% each for total viruses detected and finally Potato virus Y representing 5% of total virus detected. This survey is an _____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected]; Eyong et al.; ARRB, 36(6): 46-54, 2021; Article no.ARRB.69680 accurate and timely detection and will form the basis for mitigating yield losses resulting from virus infection on cucurbits. This is the first report of wide scale detection of viruses infecting cucurbits in Nigeria. Keywords: Cucurbitaceae; ACP-ELISA; RT-PCR; potyvirus; antisera. 1. INTRODUCTION cucumber and squash, while the USA is the main producer in the Western Hemisphere with Cucurbits represent one of the most important 4,602,370 tons [1]. Cucurbit crops are often vegetables for human consumption throughout threatened by diseases of fungal, bacterial and the world. Cucurbit species are members of the viral origin [2] and pests, such as the pumpkin family Cucurbitaceae that roughly includes 120 caterpillars (Diaphania spp.) and the melon fly genera and 800 species, which are (Bactrocera cucurbitae Coquillet), are also predominantly tropical plants though some, such serious constraints to their cultivation [3,4,5]. as four major cucurbit crops are economically Aphids, whiteflies and thrips can become serious important worldwide: melon (Cucumis melo L.), limitations to cucurbit production through the cucumber (Cucumis sativus L.), watermelon extensive damage they provoke, but also through (Citrullus lanatus (Thumb.) Mat. & Nak.), squash the transmission of viral disease [6,7,8]. At least and pumpkin (Cucurbita spp.) [1]. Global 59 viruses infecting cucurbits are known [9], production of cucurbits cultivated in the world is whose outbreaks have led to significant yield 151,212,210 tons. China is the main producer losses worldwide [6,10,11]. with 83,429,409 tons of melon, watermelon, a B C d E F g H Fig. 1 (a-h). a- Mosaic and chlorotic spot on Cucurbita pepo, b-Rugosity and mottle on Zeneria sp, c- Leaf malformation and mosaic on watermelon, d- Mosaic, green vein banding and leaf malformation on cucumber, e- leaf malformation/deformation and chlorosis on Telfairia occidental, f-Mosaic and leaf deformation on Cucurbita moschata, g-Chlorotic spots on Momordica charantia, h-Mosaic on Lagenaria siceraria 47 Eyong et al.; ARRB, 36(6): 46-54, 2021; Article no.ARRB.69680 The most commonly encountered of 2. MATERIALS AND METHODS these viruses of cucurbits are Papaya ringspot virus-W (PRSV-W), Squash leaf curl virus 2.1 Sources and Isolation of Viruses (SLCuV), Soybean mosaic virus (SMV), Squash mosaic virus (SqMV) and Squash vein Twenty-four (24) samples with typical virus-like yellowing virus (SqVYV). Others include symptoms were collected from different cucurbit Tobacco ringspot virus (TRSV), Watermelon crops, which included Telfairia occidentalis, mosaic virus (WMV), Watermelon silver Cucumis sativus, Cucurbita pepo, Lagenaria mottle virus (WSMoV), Alfalfa mosaic virus siceraria, Citrullus lanatus, Zehneria spp, (AMV), Bean pod mottle virus (BPMV), Cucumeropsis mannii, Momordica charantia and Cucurbit aphid borne yellows virus Cucurbita moschata. These cucurbits were found (CABYV), Cucurbit yellow stunting disorder growing on farms and gardens in Calabar, virus (CYSDV), Cucumber green mottle Baccocco, Akamkpa, Ehom, Akpet, Abini, Adim, mosaic virus (CGMMV), Cucumber mosaic virus Iwuru in Southern Ekori, Nko, Mkpani, Ofodua, (CMV), Melon necrotic spot virus Ofatura, Okuni, Ikom, Ugep in Central and Afrike, (MNSV), Zucchini yellow mosaic virus Alege, I gwo, Igoli, Okuku, Sankwala, Basang and (ZYMV), and Zucchini green mottle mosaic virus Udeshi in Northern Cross River State, Nigeria. (ZGMMV). These viruses have emerged as the Infected leaf samples were collected into Ziploc most economically significant viruses air tight polyethylene bags to keep them fresh to infecting cucurbits in various cucurbit-growing ensure the viability of the viruses and later regions of the globe [10]. Of these viruses CMV, transported to the Molecular Laboratory of SqMV, WMV-1 (now renamed WMV), National Institute of Horticulture Ibadan, Nigeria WMV-2, ZYMV beside SqMV which is for serology and molecular testing while the seedborne in melon are transmitted by sequencing was done at Inqaba Biotech West beetles. Others are transmitted mechanically and Africa (IBWA), Ibadan, Nigeria. by several aphid species in a non persistent manner [2]. Only very few of these viruses have been characterized and 2.2 Serological Tests identified in Nigeria. For example, [12] have reported infection of fluted pumpkin by Antigen coated plate enzyme linked Cucumber mosaic virus in northern Nigeria, immunosorbent assay (ACP-ELISA) as [13] have also characterized and reported described by [16] was used to determine the Yam mosaic virus (YMV) and Telfairia genus to which the virus isolates belonged. mosaic virus (TeMV) infecting cucurbits in Symptomatic leaf samples of 0.1g were triturated Northern Nigeria. [14] havefurther in 1mL of coating buffer (0.015M Na2 Co3 + provided a checklist of viruses prevalent in 0.0349 M NaHCo3 + dH2O) and dispensed into south west of Nigeria [15] also reported each well of ELISA plate. After incubation at infection of cucumber by Papaya Ringspot 37°C for 1h the plate was washed 3 times with virus (PRSV) and infection of Cucurbita PBS-Tween for 3 min between each wash. Cross moschata by Morroccan watermelon mosaic adsorption was made by grinding 1 g of healthy virus (MWMV) in Calabar Southern Nigeria. plant sample in 20mL of conjugate buffer (1/2 However, Information regarding large scale PBS + 0.05% Tween 20 + 0.02% egg albumin + infection of cucurbits in Southern Nigeria is 0.2% PVP). Universal potyvirus and lacking hence this study. A visit to some cucurbit Cucumovirus antisera were diluted at 1:3000 in growing farms in these areas between January to the adsorption solution and 100μL of each March 2021 revealed widespread infection of antiserum polyclonal antisera was added to wells these crops. Infected plants exhibited a wide of the ELISA plates and again incubated at 37°C range of virus-like symptoms such as for 1hour. The ELISA plates were then washed 3 mosaic, chlorotic spots, mottling, leaf times with PBS-Tween. One hundred-μL of malformation/deformation and leaf reduction, protein, A-alkaline phosphatase conjugate diluted some of which are presented in Fig. 1 (a-h). This in the ratio 1:15000 in conjugate buffer (1/2 PBS research was therefore designed to carry out a + 0.05% Tween 20 + 0.02% egg albumin + 0.2% survey on potyviruses infecting some PVP + 0 .02g NaNO3) was added per well and the cucurbits across different regions in Cross River plates incubated at 37°C for 1h. The plates were again washed 3 times with PBS-Tween. One State Nigeria, employing serological and -1 molecular tools with a view to identifying hundred -μL of 0.001g∙mL of p-nitrophenyl them. phosphate substrate in substrate buffer (97mL 48 Eyong et al.; ARRB, 36(6): 46-54, 2021; Article no.ARRB.69680 diethanolamine + 800mL H2O + 0.2g NaNO3 and min. Then 250 μL of 6M potassium acetate (pH HCl to give pH 9.8) was added per well and 6.5) were added and the tubes were transferred incubated at room temperature for 1 h. For all on ice for 20 min. After centrifugation, nucleic incubations, plates were covered with ELISA acids were precipitated with 500 μL ethanol. The cover plates to avoid edge effects and to pellet was resuspended in 50 μL RNAse free maintain uniform temperature. Healthy plant sterile water. samples were used as controls. After 1 h absorbance was measured at A405nm using an 2.4 RT-PCR ELISA plate reader (Micro Read 1000 ELISA plate analyser, U.S.A) after 1 h of incubation.